Calcium regulation of transient K+ currents. The sub-threshold activating, A-type potassium current (IA) has long been recognized as a critical component for regulating dendritic excitability and shaping back propagating action potentials. In CA1 pyramidal cells of the hippocampus IA is carried predominantly by Kv4.2 potassium channels, which are associated with a variety of accessory proteins that modulate their trafficking and kinetics. Among these interacting proteins are K-channel interacting proteins (KChIPs), small cytoplasmic proteins with four calcium-binding EF-Hand motifs and dipeptidyl-peptidase-like proteins (DPPs). IA shows a distinct functional gradient along the apical dendrites of CA1 pyramidal neurons. The reciprocal relationship between Kv4.2 and the GluN2B subunit of the N-methyl-D-aspartate receptor, and its interaction with the voltage-gated calcium channel Cav2.3 opens up the possibility of calcium dependent effects on Kv4.2 function in the synapse. Using a mutated KChIP construct that is unable to bind calcium but still interacts with Kv4.2, Jakob Gutzmann has found that changes in intracellular calcium concentration alters many Kv4.2-mediated current properties in the presence of KChIPs in a heterologous expression system. All of these effects were abolished when we used a KChIP2a construct in which its ability to bind calcium was mutated. Performing these experiments in the more physiological environment of acute hippocampal slices lead to similar results. Were now recording IA from somatic and dendritic outside-out patches, in mice order to better understand he interplay between calcium and potassium that shape CA1 pyramidal cells electrical behavior. Isomerase regulation of potassium channel trafficking and function. To identify Kv4.2 binding proteins, Jiahua Hu employed a tandem affinity purification approach to isolate the Kv4.2 protein complex from hippocampal neurons. Mass-spectrometry analysis identified known proteins such as KChIP family members and DPP6/10. The TAP-MS assay also identified an isomerase as a binding partner of Kv4.2. This binding was confirmed by brain co-immunoprecipitation, co-expression in HEK293T cell and peptide pull down in vitro. The isomerase binds to a specific Kv4.2 site, and the association is regulated by neuronal activity and seizure. The isomerizing activity may regulate Kv4.2 binding to its auxiliary subunits, suggesting it plays a role in Kv4.2 function. We are generating a knockin mouse that specifically deletes the isomerase binding site to further study the physiological function of isomerase and Kv4.2 channel. DPP6 deletion leads to memory impairments in mice We have previously shown that the Kv4 A-type K+ auxiliary subunit DPP6 plays important roles hippocampal synaptic development and function but the behavioral effects of DPP6-KO mice is unknown. Lin Lin focused on hippocampus-dependent tasks involved in recognition and learning memory. We found DPP6-KO mice to be impaired in recognition memory with a Novel Object Recognition task. In the Water Maze, DPP6-KO mice showed slow learning and impaired memory performance. In a T-Maze task, the success rate of WT mice increased over the course of the training, while DPP6-KO mice, started and finished at the same success rate without any significant learning throughout the training. Taken together, DPP6 deletion leads to behavioral impairments in recognition and spatial learning and memory. We are working on whether the behavioral deficits can be changed or rescued by DPP6 IUE microinjected in DPP6-KO mice. Loss of regulated Cav2.3 expression in a mouse model of Fragile X Syndrome Fragile X syndrome (FXS) is a severe form of intellectual disability in humans that arises from the loss of the fragile X mental retardation protein (FMRP), an mRNA binding protein that regulates translation downstream of group I metabotropic glutamate receptors (GpI mGluRs). Loss of FMRP leads to enhanced calcium spiking and neuronal excitability, thus Erin Gray sought to explore the possibility that FMRP regulates expression of the dendritic voltage gated calcium channel Cav2.3. We found previously that the cortices of FMRP KO mice have enhanced levels of Cav2.3 under basal conditions and we were curious how loss of FMRP might affect Cav2.3 expression in the hippocampus. We performed Western blotting to measure Cav2.3 protein levels from either acutely isolated or cultured hippocampal neurons and found that, similar to our previous findings in cortex, the FMRP KO have enhanced expression of Cav2.3 in the hippocampus. This increase in Cav2.3 expression should impact neuronal physiology; therefore we isolated Cav2.3 currents using whole cell electrophysiology in cultured hippocampal neurons from WT and FMRP KO mice. As predicted from our Western blot data, FMRP KO neurons exhibited greatly enhanced Cav2.3 current amplitudes. Thus, it appears that FMRP normally represses Cav2.3 translation under basal conditions and loss of FMRP leads to an increase of Cav2.3 protein in the membrane. We are also investigating the possibility that this FMRP repression of Cav2.3 expression can be regulated by upstream activity of GpI mGluRs. In support of this idea, our previous data showed that stimulation of GpI mGluRs does increase Cav2.3 translation and expression in WT neurons but not in neurons lacking FMRP. Next, to determine if FMRP is regulating translation of existing Cav2.3 mRNA or if transcription is required, we applied the transcriptional blocker Actinomycin D (ActD) to cultured neurons while stimulating GpI mGluRs. Treating neurons with ActD did not significantly inhibit the stimulation of Cav2.3 expression, suggesting that GpI mGluRs increase Cav2.3 protein by inducing translation of existing mRNA bound to FMRP. Taken together, our data suggests that FMRP represses translation of Cav2.3 under basal conditions and that activation of GpI mGluRs disinhibits FMRP to allow for active translation of Cav2.3. The loss of regulated Cav2.3 expression seen in the FMRP KO may underlie the atypical calcium signaling and neuronal excitability seen in FXS, and may be a potential therapeutic target in this disease. FMRP interacts with Cav2.3 mRNA Ying Liu has shown that Cav2.3 mRNA levels were altered in FMRP KO neurons and Cav2.3 protein levels were significantly enhanced in the FMRP KO. To study if Cav2.3 mRNA is one of the targets of FMRP, we performed RNA immunoprecipitation. Our data showed that FMRP interacts with Cav2.3 mRNA in transfected HEK293 cells and in mouse cortex and hippocampus. Our results suggest that FMRP binds to Cav2.3 mRNA directly or indirectly to repress Cav2.3 translation and regulates neuronal excitability. In related work, Jon Murphy is examining whether the dendritic FMRP regulates mRNA trafficking and protein expression of CaV2.3 and Kv4.2 in the dendrites of hippocampal neurons using the FMRP KO mouse. Recent progress has centered primarily on analysis of mRNA localization and regulation of total protein translation in neuronal dendrites of WT and FMRP knockout mouse neurons. Using fluorescence in situ hybridization to detect mRNAs for CaV2.3 and CaMKII in neurons, we have found that CaMKII mRNA, a known dendritically synthesized protein has increased abundance throughout the dendritic arbor of FMRP KO mice. FMRP is known to inhibit CaMKII translation through direct binding of CaMKII mRNA suggesting either CaMKII mRNA is more highly transcribed in FMRP knockout mice, it is no longer sequestered in mRNA-protein complexes where in situ hybridization is inhibited, or both. Conversely, CaV2.3 mRNA signals are low throughout dendrites in both WT and FMRP KO neurons. Studies of Kv4.2 mRNA localization in WT and FMRP knockout neurons are ongoing.